Correlation of photodynamic activity and singlet oxygen quantum yields in two series of hydrophobic monocationic porphyrins

Journal of Porphyrins and PhthalocyaninesJ. Porphyrins Phthalocyanines 2012; 16: 55–63

DOI: 10.1142/S1088424611004336

Published at http://www.worldscinet.com/jpp/

Copyright © 2012 World Scientific Publishing Company

INTRODUCTION

Photodynamic therapy (PDT) is a medical procedure, based on the combined action of a photosensitizer (dye) and light, that has been increasingly employed in the treatment of cancer and other skin diseases, such as psoriasis and vitiligo (leukoderma), with excellent results from the medical and aesthetic points of view. Among the several compounds, porphyrins and their derivatives have been extensively employed as photosensitizers. In fact, Photofrin® the first commercial phototherapeutic agent approved by FDA about 20 years ago, is based on hematoporphyrin oligomers, more exactly nine porphyrin units linked by ether, ester and carbon–carbon bonds [1–3]. Nevertheless, some limitations have been imposed, particularly by their long persistence time in the organism, leading to undesirable skin photosensibility that can last several weeks or even months [4–6]. For this reason, more effective and safe photosensitizers and formulations exhibiting well defined chemical compositions, higher

biocompatibility, preferential or specific accumulation in the tumor cells, faster elimination rate by the organism, lower toxicity and higher phototoxicity have been pursued [3, 7–10]. At the present time only Photofrin®, Levulan® Kerastick and Visudyne® have been approved by FDA for PDT treatment.

Although porphyrins and derivatives have interesting photodynamic properties, frequently their solubility in aqueous media is far too low for direct application in PDT treatment. A convenient strategy to increase the biocompatibility and dispersability in biological fluids is the micro and/or nanoencapsulation of water insoluble lipophilic compounds. Polymeric encapsulation is a very interesting choice for that purpose, because the capsules shell can be engineered using several biocompatible polymers and varying the degree of cross-linking [9, 11, 12], thus allowing the control of the permeability and the mechanical resistance. Also, polyethyleneglycol derivatives can be added to decrease the interaction of the polymeric capsule with the plasma components thus avoiding the loss of formulation by opsonization [13, 14] by phagocytary cells.

The binding properties of a series of meso-phenyl(N-methylpyridinium)porphyrins to vesicles and red cells

Correlation of photodynamic activity and singlet oxygen quantum yields in two series of hydrophobic monocationic porphyrins

Daiana K. Deda, Christiane Pavani, Eduardo Caritá, Maurício S. Baptista, Henrique E. Toma and Koiti Araki*◊

Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes 748, Sao Paulo, 05508-000, SP, Brazil

Received 7 May 2011Accepted 2 July 2011

ABSTRACT: The photodynamic properties of eight hydrophobic monocationic methyl and ruthenium polypyridine complex derivatives of free-base and zinc(II) meso-triphenyl-monopyridylporphyrin series were evaluated and compared using HeLa cells as model. The cream-like polymeric nanocapsule formulations of marine atelocollagen/xanthan gum, prepared by the coacervation method, exhibited high phototoxicity but negligible cytotoxicity in the dark. Interestingly, the formulations of a given series presented similar photodynamic activities but the methylated free-base derivatives were significantly more phototoxic than the respective ruthenated photosensitizers, reflecting the higher photoinduced singlet oxygen quantum yields of those monocationic porphyrin dyes.

KEYWORDS: cationic porphyrins, polymeric encapsulation, PDT, HeLa cells.

SPP full member in good standing

*Correspondence to: Koiti Araki, email: [email protected], tel: +55 11-3091-8513, fax: +55 11-3815-5579

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http://dx.doi.org/10.1142/S1088424611004336

http://dx.doi.org/10.1142/S1088424611004336

Copyright © 2012 World Scientific Publishing Company J. Porphyrins Phthalocyanines 2012; 16: 56–63

56 D. K. DEDA ET AL.

were shown to be correlated with their n-octanol/water partition coefficients (log(POW)). However, amphiphilic species exhibited significantly higher interaction and photodynamic activity [15–17] than predicted solely based on that parameter. More recently, the monocationic meso-triphenyl(3-N-methylpyiridinium)porphyrin, 3MMe, was shown to be a very efficient photosensitizer when encapsulated in atelocollagen polymeric nanocapsules prepared by coacervation method [18]. However, few are the reports [19, 20] on the influence of transition metal ions, coordinated to the porphyrin ring or to its periphery, on the photodynamic properties. Accordingly, a systematic study was carried out with two series (free-base and zinc(II) complex) of monocationic meso-triphenyl(pyridyl)porphyrin derivatives (Fig. 1) in order to assess the influence of the ring metallation and the coordination of a [Ru(bipy)2Cl]+ complex on the photodynamic properties of porphyrin dyes, using HeLa cells as model.

RESULTS AND DISCUSSION

Syntheses and characterization

The monocationic porphyrins 3MPyTPP and 4MPyTPP were prepared by the reaction of stoichiometric amounts of pyrrole, benzaldehyde and 3-pyridyl or 4-pyridylcarboxaldehyde in refluxing glacial acetic acid [24]. The small amounts of meso-tetraphenylporphyrin (TPP) and disubstituted derivatives were separated by silica-gel column chromatography using a mixture of dichloromethane and ethanol (2%) as eluent. The respective zinc(II) complexes (3- and 4-ZnMPyTPP) were obtained by refluxing them with zinc acetate in a mixture of glacial acetic acid and DMF.

3MMe and 4MMe, were obtained refluxing 3MPyTPP and 4MPyTPP with 40 times excess of methyl p-toluenesulfonate in dimethylformamide (DMF), purified by neutral alumina column chromatography and precipitated as chloride salts in saturated NaCl solution to increase the biocompatibility and solubility in aqueous media. The Zn-3MMe and Zn-4MMe were prepared in a similar way.

The series of ruthenated porphyrins (3MRu, 4MRu, Zn-3MRu and Zn-4MRu) were obtained by reacting 3MPyTPP, 4MPyTPP, Zn-3MPyTPP and Zn-4MPyTPP with about 1% excess of the [Ru(bipy)2Cl(H2O)]NO3 complex, in a CH2Cl2/DMF 4:1 v/v mixture, for 60 min [17] and purified by neutral alumina column chromatography, using a mixture of CH2Cl2 and ethanol as eluent.

The methylated and ruthenated free-base porphyrin series exhibited characteristic UV-vis spectral profiles with an intense band around 420 nm (Soret band) and four less intense Q-bands at 515, 565, 596 and 650 nm (Fig. 2). A bipyridine pp→pp* internal transition band and a broad RuII(dp)→bipy(pp*) MLCT band were also observed in the ruthenated derivatives, respectively at 295 and 470 nm. A similar behavior was observed for the Zn(II) porphyrin series, but the Soret band was shifted to 428 nm and only two Q-bands were observed at 560 and 610 nm.

The two series of methylated and ruthenated amphiphilic monocationic porphyrins (Fig. 1) were characterized by 1H NMR. The b-pyrrole protons of the para-isomers were found as a broadened singlet (8H) between 8.4 and 8.9 ppm, while the inner ring protons appeared as a singlet (2H) at -2.8 ppm. The pyridyl protons were found as a pair of doublets at 8.0 and 9.7 ppm, while the phenyl group protons were

Fig. 1. Scheme showing the structure of the eight monocationic meso-triphenyl(pyridyl)porphyrin derivatives, highlighting the structure of 3MMe

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CORRELATION OF PHOTODYNAMIC ACTIVITY 57

found as a double doublet and a multiplet at 7.6 and 8.1 ppm. The protons of the bipyridyl ligands appeared as very characteristic signals in the 7 to 10.1 ppm range. As expected, an intense singlet (3H) was observed at 4.6 ppm in the N-methylated derivatives. The 1H NMR signals of the [Ru(bipy)2Cl]+ derivatives of the porphyrin para-isomers were sharper and better resolved than the signals of the new meta-isomers, but the integration was always consistent with the expected number of protons. Accordingly, the presence of a [Ru(bipy)2Cl]+ group bond to the periphery of the porphyrin ring was further confirmed by cyclic voltammetry. In fact, the characteristic reversible pair of voltammetric waves at 0.9 V, assigned to the RuIII/RuII redox process, had the same intensity of the monoelectronic reversible reduction processes of the porphyrin ring at -0.9 and -1.3 V, thus confirming the presence of a ruthenium complex coordinated to the porphyrin ring. The irreversible process observed at +1.38 V was assigned to the oxidation of the porphyrin ring.

Singlet oxygen quantum yields (fD)

Meso-arylporphyrins are type II photosensitizers whose photodynamic activity depend on the formation of singlet oxygen by energy-transfer from electronically excited dye molecules to dioxygen [1–3, 6, 25], upon irradiation with light of suitable wavelength [2, 5, 6, 26, 27]. The photoinduced singlet oxygen quantum yields (fD) of all porphyrin dyes were evaluated from the slope of the 1Dg(O2) emission intensity as a function

of the laser power plot. The absorbance at 532 nm of the eight porphyrin dyes and the TPP standard in CH2Cl2 solution was adjusted to 0.02 a.u., and the power of the laser pulses varied in the 1 to 8 mJ.cm-2 range. A typical set of decay curves, used to collect the emission intensity at 1270 nm for the 4MRu and the Zn-4MRu derivatives, are shown in Fig. S1 (see Supporting information section). The phosphorescence intensities were measured 70 ms after the laser pulse and plotted as a function of the corresponding laser power. Linear correlations were found for both porphyrin series and fD determined from the ratio of their slopes with that found for the TPP standard, in the same experimental conditions. The free-base porphyrin derivatives exhibited significantly higher 1Dg(O2) phosphorescence than the corresponding zinc(II) porphyrin derivatives, reflecting their higher energy transfer quantum efficiencies. The fD values, the lifetimes (1/k) and the photodynamic efficiencies (% of cell death) for the eight porphyrin dyes are listed in Table 1.

The lifetime of 1Dg(O2) defines the maximum radius of action of this reactive species. Values in the 140–180 ms range were measured in CH2Cl2, which are consistent with the lifetime of singlet oxygen in hydrophobic environments. In fact, it can be as high as 200 ms in organic solvents in the absence of quenchers, but decreases to 2–4 ms in aqueous solution [28]. In the cytosol, however, the lifetime decreases to about 10–50 ns, such that the perimeter of action is reduced to about 0.1 mm from the point where it is generated.

Fig. 2. Absorption spectra of (a) 3MMe and Zn-3MMe, (b) 4MMe and Zn-4MMe, (c) 3MRu and Zn-3MRu, and (d) 4MRu and Zn-4MRu in CH2Cl2. Inset: exploded view in the visible range

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The free-base porphyrins, particularly the 3MMe and 4MMe derivatives, showed higher fD and photodynamic efficiencies than the respective zinc(II) porphyrins. Also, the ruthenium complex is contributing to diminish further the 1Dg(O2) quantum yields of the macrocyle, possibly introducing new competing relaxation pathways for the excited state species.

Toxicity and phototoxicity of the monocationic porphyrins

Coacervation is one of the most commonly used methods for the preparation of nanocapsules using natural polymeric materials such as sodium alginate and gelatin. The procedure involves two steps: (a) the o/w emulsification by mixing an organic phase containing the active substances with an aqueous polymeric phase by mechanical stirring and/or ultrasound processing; followed by (b) the coacervation process that can be induced by addition of electrolytes, decrease of temperature or dehydration agents [21, 29, 30].

The delivery efficiency of a photosensitizer [11, 31] to cells by micro/nanocapsule formulations are mainly controlled by shell properties [21, 32] such as morphology, size, mechanical strength and permeability, which generally are defined by the chemical composition, temperature, pressure and pH, that may also influence the cell viability, cytolocalization and interaction with the blood components, as described by Dash et al. [33].

The photodynamic properties of a dye depend on three major factors in addition to the pharmacokinetic properties and biodistribution: (a) their ability to interact with cells and tissues, (b) the quantum efficiency of the photosensitizer, and (c) the mechanism of the

photodynamic action. The first two are strongly dependent on the molecular structure, such that the affinity was found to be a function of the lipophilic character of the cationic dyes. However, this is abnormally enhanced in the case of amphiphilic species [16] that penetrate more deeply into the cell membrane as a consequence of the unique spatial distribution of lipophilic and hydrophilic groups in those molecules. Also, amphiphilic Zn(II) porphyrins was shown to bind preferentially to the cell membrane, improving the PDT efficiency [20]. The meso-pyridylporphyrins are type II photosensitizers, acting essentially through energy transfer to molecular oxygen and production of singlet oxygen, rather than the direct electron transfer from the excited species to biomolecules. The coordination of zinc(II) [34, 35] was shown to improve the PDT efficiency because tend to enhance the type II character of the photosensitizer. In fact, tetraruthenated porphyrins [36], particularly the zinc(II) derivative, showed to be a better type II photosensitizer than methylene blue. A similar behavior was observed for N-confused porphyrins and its Ag(III) complex [37].

The two series of monocationic porphyrins have very low solubility in aqueous media such that they can cause serious problems when injected intravenously due to precipitation and clogging of capillary veins. Unfortunately, the affinity of the dye for tumor cells and tissues is proportional to log(POW) such that high affinity usually means low solubility in biological fluids, hindering their direct use in PDT. This problem can be overcome by encapsulating them in micro and nanocapsules with appropriate surface functional groups and surface charge [11, 12].

In addition, the composition and size can be controlled to impart biocompatibility and optimal

Table 1. Fluorescence quantum yields, ffl, singlet oxygen 1Dg(O2) quantum yields, fD, lifetimes t (in CHCl3 solution) and photodynamic efficiencies fPD towards HeLa cells of the eight monocationic porphyrin dyes and TPP, used as standard

Porphyrin ffl (SD) fD (SD) t, ms (SD) fPD, %

TPP 0.150 0.50 183.4 (4.2)

3MMe 0.3431 (0.0086) 0.79 (0.04) 163.2 (3.1) 92.3

4MMe 0.4583 (0.0030) 0.55 (0.02) 160.2 (2.6) 92.3

3MRu 0.0210 (0.0007) 0.50 (0.02) 161.1 (4.7) 71.9

4MRu 0.0297 (0.0006) 0.51 (0.03) 162.6 (1.4) 74.9

Zn-3MMe 0.0124 (0.0003) 0.40 (0.01) 140.6 (7.5) 54.0

Zn-4MMe 0.0372 (0.0015) 0.48 (0.03) 138.9 (5.0) 54.5

Zn-3MRu 0.0148 (0.0002) 0.40 (0.03) 139.3 (2.6) 53.0

Zn-4MRu 0.0249 (0.0003) 0.36 (0.02) 144.7 (3.4) 58.8

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interaction and delivery of their content to malignant cells. For this purpose, polymeric formulations were prepared by encapsulating the two series of monocationic porphyrins in marine athelocolagen/xantam gum polymeric microcapsules, that were reprocessed using a high power ultrasonic tip monitoring the size by DLS [18]. Formulations with the largest fraction of capsules in the 400–500 nm range and histograms exhibiting bimodal size distribution were obtained (see Supporting information section Fig. S2). The porphyrin concentration was 1.0 × 10-4 M in all formulations. The cytotoxicity was evaluated by incubating 1.0 × 105 HeLa cells with 1.0 mL of a 1 mM porphyrin formulation (in colorless DMEM culture media) for 6 h, in the dark.

When compared to the dark control experiments in the absence of any sensitizer, the dark toxicity of all sensitizers investigated was negligible (Fig. S3). However, the cell viability decreased dramatically when the cells, incubated with the free-base porphyrin formulations, were irradiated with the 650 nm laser, in contrast with the control (Fig. 3). Note that the 4MMe and 3MMe formulations reduced the cell viability in more than 80%, while a 50–60% decrease was found for 3MRu and 4MRu. In fact, the N-methylated derivatives consistently showed a much higher photodynamic efficiency than the N-ruthenated derivatives. The localization of the peripheral groups in the para- or meta-position had no significant influence on the photodynamic activity within experimental error, showing that the solubility and binding affinity of those photosensitizers to the HeLa cells are virtually the same for both isomers.

At this point, it is interesting to remember that the fluorescence quantum yield of 4MPyTPP in CH2Cl2 is ffl(lexc 512 nm) = 0.099, only slightly smaller than for TPP standard ((ffl(lexc 512 nm) = 0.11). The coordination of a [Ru(bipy)2Cl]+ group to the pyridyl N-atom did not change the fluorescence spectra profile (two bands

at 655 and 715 nm) indicating that the lowest excited state in 3MRu and 4MRu is localized in the porphyrin ring and not in the peripheral [Ru(bipy)2Cl(pyP)]+ complex. However, the fluorescence quantum yield decreased almost two orders of magnitude to 2.9 × 10-3 suggesting that other relaxation pathways, such as the non-radiative decay and the intersystem crossing, were favored by coordination of the ruthenium complex (Fig. S4). Interestingly, the photodynamic efficiency was reduced only about 20% suggesting that the energy transfer from the excited porphyrin to dioxygen should be much faster than the fluorescence emission and other pathways, including the non-radiative decay. However, considering that the lifetime of ruthenated porphyrins in the singlet excited state should be much smaller than that for the starting MPyTPP (~5 ns in CH2Cl2), there is not enough time for the occurrence of efficient bimolecular processes. Thus, the energy transfer to dioxygen should be taking place after intersystem crossing to the excited triplet state. In this case, the decreased of the fluorescence quantum yield can be explained by the increase of the intersystem-crossing rate induced by the coordination of the ruthenium complex. Analogous behavior was previously reported for the doubly N-confused Ag(III) complex. [37]

Unfortunately, the series of monocationic zinc(II) porphyrins have the lowest energy absorption band around 610 nm (Fig. 2) and can not be excited by the red laser (lem = 650 nm) used for the free-base series. In order to get comparable results with both series, experiments were carried out using a mercury lamp as white light source. This presents a broad emission band in the visible range that can be used to excite both, the free-base and the zinc(II) porphyrin derivatives.

Accordingly, experiments were carried out as described above for the free-base porphyrin derivatives with both series of porphyrin dyes, except for the irradiation with a white mercury lamp source (16 mW.cm-2) for an hour. About 55% decrease in cell viability was found for all zinc(II) porphyrin series (Fig. 4a), while the free-base series once again showed distinct photodynamic behavior for the methylated (90% decrease) and ruthenated (70% decrease) derivatives (Fig. 4b). These results are consistent with those described above using the red laser as light source (Fig. 3), where no significant differences were observed in the photodynamic properties of the para- and the meta-isomers. This result is also consistent with a previous report in which a convergence of the log(POW) values was shown for both mono(N-methylpyridinium)porphyrin derivatives, while increasing differences were observed as the number of N-methylpyridinium groups increased [16].

Similar results were obtained for the zinc(II) porphyrin series suggesting that the coordination of Zn(II) ion to the porphyrin ring contributed to enhance the intersystem crossing rate alike the coordination of the [Ru(bipy)2Cl]+ complex to the pyridyl N-atom. Furthermore, the energy

Fig. 3. Phototoxicity of the monocationic free-base porphyrin derivatives in polymeric nanocapsules (1 mM) towards HeLa cells. The cells (1 × 105) were incubated for 6 h and irradiated with a 650 nm laser source for 10 min

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of the Zn(II) porphyrin excited triplet state should be lower than the charge transfer excited state localized in the ruthenium complex, because otherwise the photodynamic efficiency should be significantly decreased by energy transfer. In addition, electron transfer processes should not be taking place since they would decrease further the photodynamic activity.

The fD and photodynamic efficiency of the porphyrin derivatives are compared in Fig. 5. The highest phototoxicity observed for 3MMe can be assigned to its higher 1Dg(O2) quantum yield. Interestingly, the photodynamic activity of 4MMe was much higher than that expected based on the fD suggesting that the value was underestimated, probably as a consequence of aggregation even at the low concentrations used in the measurements [15]. Likewise, the lower photodynamic efficiency observed for zinc(II) porphyrin and ruthenium complex derivatives can be assigned to their much lower 1Dg(O2) quantum yields.

EXPERIMENTAL

Synthesis of the monocationic porphyrin derivatives

3MPyTPP and 4MPyTPP. The 5,10,15-triphenyl-20-(3-pyridyl)porphyrin, 3MPyTPP, and the 5,10, 15-triphenyl-20-(4-pyridyl)porphyrin, 4MPyTPP, were synthesized and characterized according to a previously described method [15, 17]. A mixture of 53 mmol of benzaldehyde and 18 mmol of 3-pyridyl carboxaldehyde (or 4-pyridyl carboxaldehyde) was reacted with 71 mmol of pyrrol in glacial acetic acid, in the dark. After 2 h, the solvent was removed in a flash evaporator and the porphyrin precipitated-out in a mixture of ethanol and N,N-dimethylformamide (9:1 v/v). The solid was filtered, washed with the same mixture of solvents, dried and purified by silica-gel column chromatograph using suitable mixtures of CH2Cl2/EtOH as eluent. Yield 11%. (3MPyTPP). UV-vis (CH2Cl2): lmax, nm (log e)

Fig. 4. HeLa cell viability after incubation with monocationic porphyrins incorporated in polymeric nanocapsules, in the dark and after irradiation for 1 h with a 16 mW.cm-2 white mercury lamp. (a) Zn(II) and (b) free-base porphyrin derivatives

Fig. 5. Photodynamic efficiency and respective 1Dg(O2) quantum yields of the eight monocationic porphyrin derivatives encapsulated in polymeric nanocapsules, when irradiated with a 16 mW.cm-2 white mercury lamp

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418 (5.68), 515 (4.32), 548 (3.95), 590 (3.83) and 650 (3.63). 1H NMR (500 MHz, DMSO, TMS): d, ppm 9.74 (m, 1H), 9.39 (d, 1H), 8.98 (m, 1H), 8.83 (d, 2H), 8.79 (s, 4H), 8.72 (d, 2H), 8.46 (m, 1H), 8.15 (m, 6H), 7.71 (m, 9H), -2.82 (s, 2H). (4MPyTPP). UV-vis (CH2Cl2): lmax, nm (log e) 417 (5.70), 515 (4.28), 550 (3.92), 590 (3.80) and 650 (3.61). 1H NMR (500 MHz, DMSO, TMS): d, ppm 8.96 (m, 2H), 8.83 (d, 2H), 8.79 (s, 4H), 8.73 (d, 2H), 8.15 (m, 6H), 8.11 (m, 2H), 7.71 (m, 9H), -2.80 (s, 2H).

Preparation of the free-base porphyrin series3MMe and 4MMe. The monopyridylporphyrins

3MPyTPP and 4MPyTPP were refluxed with 40-fold excess of methyl p-toluenesulfonate in N,N-dimethylformamide (DMF, 4 h), concentrated in a rotary evaporator and poured into a saturated NaCl aqueous solution. The precipitate was separated by filtration, redissolved in DMF and precipitated again in saturated NaCl solution to obtain the N-methylated porphyrins as chloride salts. After repeating this process three more times, the N-methylated dyes were purified by alumina column chromatography using a mixture of CH2Cl2 and EtOH as eluent. Yield 87%. (3MMe). UV-vis (CH2Cl2): lmax, nm (log e) 422 (5.06), 515 (3.95), 550 (3.59), 590 (3.55) and 650 (3.45). 1H NMR (500 MHz, DMSO, TMS): d, ppm 10.02 (s, 1H), 9.50 (d, 1H), 9.40 (d, 1H), 9.04 (s, 2H), 8.90 (m, 6H), 8.58 (t, 1H), 8.23 (m, 6H), 7.86 (m, 9H), 4.64 (s, 3H), -2.85 (s, 2H). (4MMe). UV-vis (CH2Cl2): lmax, nm (log e) 422 (5.17), 515 (4.07), 550 (3.78), 590 (3.60) and 650 (3.58). 1H NMR (500 MHz, DMSO, TMS): d, ppm 9.22 (m, 2H), 8.87 (d, 2H), 8.82 (s, 4H), 8.69 (d, 2H), 8.24 (m, 2H), 8.15 (m, 6H), 7.73 (m, 9H), 4.62 (s, 3H), -2.81 (s, 2H).

3MRu and 4MRu. The ruthenated free-base porphyrin derivatives were obtained by the reaction of 3MPyTPP and 4MPyTPP with [Ru(bipy)2Cl(H2O)]NO3

prepared just before use. Typically, 85 mg of AgNO3 was dissolved in 5 mL of DI-water and added to 236 mg of [Ru(bipy)2Cl2] dissolved in 15 mL of a 2:1 DMF/ethanol mixture, stirred for 20 min at 50 °C, and filtered through a Celite layer. The filtrate was concentrated to 5 mL, mixed with an equal volume of CH2Cl2 and reacted with 60 mg of the respective porphyrins, dissolved in 10 mL of CH2Cl2/DMF 4:1 mixture. After completion of the reaction (~20 min, 50 °C), the solvent was removed in a rotary evaporator, the solid dissolved in ~5 mL of DMF and added into an aqueous lithium trifluoromethanesulphonate (LiCF3SO3) solution. The ruthenated porphyrins, 3MRu and 4MRu, were pre-cipitated (CF3SO3

- salts) and isolated as dark brown solids after filtration, washing with water and drying in a desiccator under vacuum. The compounds were finally purified by alumina column chromatography using a mixture of CH2Cl2 and ethanol as eluent. Yield ~90%. (3MRu). UV-vis (CH2Cl2): lmax, nm (log e) 294 (4.70), 417 (5.60), 520 (4.20), 550 (4.13), 588 (3.95) and 650 (3.77). 1H NMR (500 MHz, DMSO, TMS): d, ppm 10.13

(s, 1H), 9.45 (s, 1H), 9.10–8.65 (10H), 8.60–8.45 (4H), 8.35(m, 3H), 8.28–7.85 (13H); 7.80–7.40 (13H), 7.05(m, 1H), -2.78 (s, 2H). (4MRu). UV-vis (CH2Cl2): lmax, nm (log e) 293 (4.80), 417 (5.70), 520 (4.30), 552 (4.16), 588 (3.88) and 649 (3.70). 1H NMR (500 MHz, DMSO, TMS): d, ppm 10.19 (m, 1H), 8.93 (m, 1H), 8.80 (m, 8H), 8.59 (m, 2H), 8.59 (m, 1H), 8.48 (m, 1H), 8.32 (m, 1H), 8.25 (m, 1H), 8.12 (m, 6H), 8.12 (m, 2H), 8.02 (m, 2H), 8.01 (m, 1H), 7.80 (m, 1H), 7.68 (m, 9H), 7.62 (m, 1H), 7.35 (m, 1H), 7.10 (m, 1H), -2.80 (s, 2H).

Preparation of the zinc(II) porphyrin seriesZn(II) porphyrin derivatives. The zinc(II)

porphyrins, Zn-3MPyTPP and Zn-4MPyTPP, were obtained by refluxing 3MPyTPP and 4MPyTPP with zinc acetate in a mixture of glacial acetic acid and DMF (yield ~90%). Then, the N-methylpyridinium (Zn-3MMe and Zn-4MMe) and the ruthenium complex derivatives (Zn-3MRu and Zn-4MRu) were obtained with ~90% yield, using the procedures described above for the preparation of the respective free-base porphyrin derivatives. (Zn-3MMe). UV-vis (CH2Cl2): lmax, nm (log e) 429 (5.07), 560 (3.84) and 600 (3.40). 1H NMR (500 MHz, DMSO, TMS): d, ppm 10.05 (s, 1H), 9.37 (d, 1H), 9.32 (d, 1H), 8.96 (s, 2H), 8.83 (m, 6H), 8.55 (t, 1H), 8.17 (m, 6H), 7.84 (m, 9H), 4.69 (s, 3H). (Zn-4MMe). UV-vis (CH2Cl2): lmax, nm (log e) 428 (4.90), 560 (3.90) and 610 (3.33). 1H NMR (500 MHz, DMSO, TMS): d, ppm 9.18 (m, 2H), 8.82 (d, 2H), 8.61 (s, 4H), 8.39 (d, 2H), 8.19 (m, 2H), 8.10 (m, 6H), 7.67 (m, 9H), 4.67 (s, 3H). (Zn-3MRu). UV-vis (CH2Cl2): lmax, nm (log e) 295 (4.88), 427 (5.52), 560 (3.97) and 600 (3.70). 1H NMR (500 MHz, DMSO, TMS): d, ppm 9.83 (s, 1H), 9.22 (d, 1H), 9.00–8.3 (13H), 8.25–8.00 (7H), 8.00–7.70 (9H); 7.60–7.35 (14H), 6.85 (m, 1H). (Zn-4MRu). UV-vis (CH2Cl2): lmax, nm (log e) 295 (4.76), 427 (5.45), 552 (3.02) and 603 (2.92). 1H NMR (500 MHz, DMSO, TMS): d, ppm 9.97 (m, 1H), 8.90 (m, 1H), 8.76 (m, 8H), 8.61 (m, 1H), 8.61 (m, 2H), 8.41 (m, 1H), 8.31 (m, 1H), 8.23 (m, 1H), 8.11 (m, 2H), 8.11 (m, 6H), 8.01 (m, 1H), 8.01 (m, 2H), 7.80 (m, 1H), 7.66 (m, 3H), 7.63 (m, 9H), 7.60 (m, 1H), 7.12 (m, 1H), 6.95 (m, 1H).

Preparation of the polymeric nanocapsule formulations

The eight porphyrin derivatives were encapsulated using the coacervation method, as described previously [18]. Typically, 1.0 mL of a porphyrin derivative solution (3.0 mM in CH2Cl2) was dispersed in a mixture of isopropylmyristate, almond oil and Tween 20 (1.2% v/v) using a Turrax, and poured into an aqueous xanthan gum suspension. Finally, marine atellocollagen and sodium sulfate were added under vigorous stirring to prepare a stable cream-like formulation of polymeric microcapsules [21], with the porphyrin derivatives dissolved in the oily core. The concentration of the dye was adjusted to 1 × 10-4 M. The average size of the capsules was diminished to 200–400 nm using an ultrasonic tip (750 W,

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VibraCell, from Sonics), monitoring the size distribution by dynamic light scattering in a Microtrac Inc. Nanotrac 252 equipment.

Toxicity and phototoxicity of the nanocapsule formulations

The toxicity and phototoxicity of the monocationic porphyrin dyes were evaluated using human cervical epithelioid carcinoma cells (HeLa) as model. They were cultivated in DMEM culture media, containing 10% of bovine fetal serum and 1% of peniciline/streptomicine, in a Thermo Electron Corporation HEPA class 100 incubator, at 37 °C and atmosphere with 5% of CO2. Then, 1 × 105 cells were transferred to 2.0 cm diameter culture plates with 12 wells and incubated for 15 h for cell adhesion. Then, the culture media was removed and 1.0 mL of a 100 times diluted emulsion ([porphyrin] = 1 mM) was added and incubated in the same conditions as described above.

The toxicity of the porphyrin dye formulations were evaluated by the MTT method after removal of the culture media containing the porphyrin formulation, careful washing of the wells with saline phosphate buffer (PBS) and addition of 1.0 mL of colorless DMEM culture media. The number of viable cells was estimated 14 h after by measuring the ratio of the absorbance at 550 nm found for the wells incubated with and without (control experiment, 100% viability) a porphyrin formulation, using an Infinite M200 ELISA reader (TECAN). The absorbances are proportional to the amount of formazan crystals [22] formed after incubation of the cells with a 2.0 mg.mL-1 methylthiazyldiphenyltetrazolium bromide (MTT) solution in PBS for 2 h, careful removal of the excess of the green aqueous supernatant solution and solubilization of the crystals in 1.0 mL of DMSO.

The phototoxicity was evaluated in the same way but after irradiation of the adhered cells with a 650 nm laser (LASERLine, 70 mW.cm-2, free-base series) for 10 min (periodic pulses of 1 min with 1 min intervals); or with a white light source (tungsten/halogen lamp, 16 mW.cm-2, zinc(II) porphyrin series) for 60 min. The results are the average of at least two independent experiments performed in triplicate to ensure the reproducibility of the experimental conditions.

Singlet oxygen quantum yields (fD)

The singlet oxygen (1Dg(O2)) quantum yields (fD) of the two series of monocationic porphyrin derivatives were determined from the ratio of the slopes of the phosphorescence intensity at 1270 nm vs. the laser power plots, using meso-tetraphenylporphyrin, TPP, as standard (fD(1O2) = 0.5) [23]. The 1Dg(O2) phosphorescence emission decay curves were registered using a time-resolved Edinburg Analytical Instruments NIR flash-photolysis instrument equipped with a Nd:YAG Continuum Surelite III pulsed laser (l = 532 nm, 10 Hz),

a liquid nitrogen cooled (-80 °C) Hamamatsu R5509 photomultiplier and LP900 aquisition software. The emitted light was passed through a silicon filter and a monochromator before reaching the NIR-PMT. The absorbance at 532 nm of the porphyrin derivatives in chloroform solution was adjusted to about 0.02 a.u. (~1 mM), and the phosphorescence intensity measured 10 ns after the laser pulse.

CONCLUSION

Two series of monocationic porphyrin derivatives were synthesized and their photodynamic activity evaluated using HeLa cells as model, after incorporation in polymeric marine atelocollagen/xanthan gum nanocapsules. The methylated free-base porphyrin derivatives exhibited higher phototoxicity inactivating 92% of the cells after 60 min of irradiation with a white light source, but no significant differences were observed for the para and meta isomers. The activity of the ruthenated porphyrins were ~20% lower, but was diminished even further by the coordination of Zn(II) ion to the porphyrin ring. In fact, all Zn(II) porphyrin derivatives exhibited similar phototoxicity and inactivate ~50% of the HeLa cells. Those results showed a correlation with their singlet oxygen quantum yields, such as in the case of the Zn(II) porphyrin derivatives that exhibited the lowest photodynamic activities and fD values. In conclusion, the meta and para isomers of the methylated free-base monocationic porphyrins exhibited the highest photodynamic efficiencies and are promising candidates as PDT photosensitizers.

Acknowledgements

We gratefully acknowledge the financial support from the Brazilian agencies, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Supporting information

Singlet oxygen fluorescence decay curves, polymeric nanocapsules size distribution histograms and toxicity in the dark and fluorescence spectra of free-base monocationic porphyrins, (Figs S1–S4) are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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Correlation of photodynamic activity and singlet oxygen quantum yields in two series of hydrophobic monocationic porphyrins

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